Farah Lizotte

Increased SHP-1 Protein Expression by High Glucose Levels Reduces Nephrin Phosphorylation in Podocytes

Background: Elevated expression of SHP-1 in diabetic podocytes may potentially interact with nephrin phosphorylation. Results: Rat nephrin Tyr1127 and Tyr1152 are essential for SHP-1 induced nephrin dephosphorylation under high-glucose conditions. Conclusion: SHP-1 contributes to nephrin deactivation in podocytes exposed to high glucose levels. Significance: Reduction of SHP-1 may serve as potential therapeutic target to prevent glomerular pathology in diabetes. Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr1127 and Tyr1152 are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2+/C96Y) compared with control littermate mice (Ins2+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.

PKCδ Impaired Vessel Formation and Angiogenic Factor Expression in Diabetic Ischemic Limbs

Decreased collateral vessel formation in diabetic peripheral limbs is characterized by abnormalities of the angiogenic response to ischemia. Hyperglycemia is known to activate protein kinase C (PKC), affecting the expression and activity of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The current study investigates the role of PKCδ in diabetes-induced poor collateral vessel formation and inhibition of angiogenic factors expression and actions. Ischemic adductor muscles of diabetic Prkcd+/+ mice exhibited reduced blood reperfusion, vascular density, and number of small vessels compared with nondiabetic Prkcd+/+ mice. By contrast, diabetic Prkcd−/− mice showed significant increased blood flow, capillary density, and number of capillaries. Although expression of various PKC isoforms was unchanged, activation of PKCδ was increased in diabetic Prkcd+/+ mice. VEGF and PDGF mRNA and protein expression were decreased in the muscles of diabetic Prkcd+/+ mice and were normalized in diabetic Prkcd−/− mice. Furthermore, phosphorylation of VEGF receptor 2 (VEGFR2) and PDGF receptor-β (PDGFR-β) were blunted in diabetic Prkcd+/+ mice but elevated in diabetic Prkcd−/− mice. The inhibition of VEGFR2 and PDGFR-β activity was associated with increased SHP-1 expression. In conclusion, our data have uncovered the mechanisms by which PKCδ activation induced poor collateral vessel formation, offering potential novel targets to regulate angiogenesis therapeutically in diabetic patients.

Persistent Insulin Resistance in Podocytes Caused by Epigenetic Changes of SHP-1 in Diabetes.

Poor glycemic control profoundly affects protein expression and the cell signaling action that contributes to glycemic memory and irreversible progression of diabetic nephropathy (DN). We demonstrate that SHP-1 is elevated in podocytes of diabetic mice, causing insulin unresponsiveness and DN. Thus, sustained SHP-1 expression caused by hyperglycemia despite systemic glucose normalization could contribute to the glycemic memory effect in DN. Microalbuminuria, glomerular filtration rate, mesangial cell expansion, and collagen type IV and transforming growth factor-β expression were significantly increased in diabetic Ins2+/C96Y mice compared with nondiabetic Ins2+/+ mice and remained elevated despite glucose normalization with insulin implants. A persistent increase of SHP-1 expression in podocytes despite normalization of systemic glucose levels was associated with sustained inhibition of the insulin signaling pathways. In cultured podocytes, high glucose levels increased mRNA, protein expression, and phosphatase activity of SHP-1, which remained elevated despite glucose concentration returning to normal, causing persistent insulin receptor-β inhibition. Histone posttranslational modification analysis showed that the promoter region of SHP-1 was enriched with H3K4me1 and H3K9/14ac in diabetic glomeruli and podocytes, which remained elevated despite glucose level normalization. Hyperglycemia induces SHP-1 promoter epigenetic modifications, causing its persistent expression and activity and leading to insulin resistance, podocyte dysfunction, and DN.

Combination of high-fat/high-fructose diet and low-dose streptozotocin to model long-term type-2 diabetes complications

The epidemic of type 2 diabetes mellitus (T2DM) is fueled by added fructose consumption. Here, we thus combined high-fat/high-fructose diet, with multiple low-dose injections of streptozotocin (HF/HF/Stz) to emulate the long-term complications of T2DM. HF/HF/Stz rats, monitored over 56 weeks, exhibited metabolic dysfunctions associated with the different stages of the T2DM disease progression in humans: an early prediabetic phase characterized by an hyperinsulinemic period with modest dysglycemia, followed by a late stage of T2DM with frank hyperglycemia, normalization of insulinemia, marked dyslipidemia, hepatic fibrosis and pancreatic β-cell failure. Histopathological analyses combined to [18F]-FDG PET imaging further demonstrated the presence of several end-organ long-term complications, including reduction in myocardial glucose utilization, renal dysfunction as well as microvascular neuropathy and retinopathy. We also provide for the first time a comprehensive µ-PET whole brain imaging of the changes in glucose metabolic activity within discrete cerebral regions in HF/HF/Stz diabetic rats. Altogether, we developed and characterized a unique non-genetic preclinical model of T2DM adapted to the current diet and lifestyle that recapitulates the major metabolic features of the disease progression, from insulin resistance to pancreatic β-cell dysfunction, and closely mimicking the target-organ damage occurring in type 2 diabetic patients at advanced stages.

Deletion of AT2 Receptor Prevents SHP-1–Induced VEGF Inhibition and Improves Blood Flow Reperfusion in Diabetic Ischemic HindlimbHighlights

Objective— Ischemia caused by narrowing of femoral artery is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes mellitus. We have previously reported that the inhibition of the angiogenic response to VEGF (vascular endothelial growth factor) in diabetic mice was associated with the increased expression of SHP-1 (SH2 domain–containing phosphatase 1), a protein that can be activated by the AT2 (angiotensin II type 2) receptor. Deletion of AT2 receptor has been shown to promote angiogenesis within the ischemic muscle. However, the relative impact of AT2 receptor in diabetic condition remains unknown. Approach and Results— Nondiabetic and diabetic AT2 null (Atgr2−/Y) mice underwent femoral artery ligation after 2 months of diabetes mellitus. Blood perfusion was measured every week ⩽4 weeks post-surgery. Expression of the VEGF, SHP-1, and renin–angiotensin pathways was evaluated. Blood flow in the ischemic muscle of diabetic Atgr2−/Y mice recovered faster and ⩽80% after 4 weeks compared with 51% recovery in diabetic control littermates. Diabetic Atgr2−/Y had reduced apoptotic endothelial cells and elevated small vessel formation compared with diabetic Atgr2+/Y mice, as well as increased SHP-1 expression and reduced VEGF receptor activity. In endothelial cells, high glucose levels and AT2 agonist treatment did not change SHP-1, VEGF, and VEGF receptor expression. However, the activity of SHP-1 and its association with the VEGF receptors were increased, causing inhibition of the VEGF action in endothelial cell proliferation and migration. Conclusions— Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.